The primary objective of the proposed protocol is to more clearly define the mechanism(s) of urinary acidification by the direct determination of pH, pCO2, and total CO2 concentration in vivo in the accessible nephrons of the rat kidney utilizing standard cortical and papillary micropuncture techniques. In this series of studies, three specific areas will be investigated: (1) determination of the magnitude and direction of the disequilibrium pH in proximal and distal convoluted tubules, (2) contribution of luminal carbonic anhydrase to bicarbonate reabsorption in the in vivo perfused isolated proximal tubule, and (3) determination of pH, pCO2, and total CO2 concentration in the accessible segments of the renal papilla. These studies will utilize pH and pCO2 microelectrodes developed in our laboratory for application in rat micropuncture, as well as the microcalorimetric method of Vurek for determination of total CO2 concentration. These studies should allow differentiation between the two proposed mechanisms of bicarbonate reabsorption: 1) H plus secretion, or 2) primary HCO minus 3 reabsorption. In summary, the proposed studies will utilize microelectrode and microcalorimetric techniques in concert to evaluate acid-base homeostasis directly by application of cortical and papillary micropuncture and in vivo microperfusion techniques.